Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells

1 Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF)alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2 Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml(-1)) and TNF alpha (100 u ml(-1)) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3 Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4 Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5 We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited by anti-VCAM-1. 6 Sodium orthovanadate had no effect on TNF alpha-induced U937 adhesion but dose-dependently enhanced adhesion to unstimulated HUVEC. Vanadate-induced adhesion was inhibited by an antibody against VCAM-1. 7 These results demonstrate that PTK-mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine-induced adhesion molecule expression.